The antibacterial action of cloths and sanitizers and the use of environmental alternatives in food industries.

Introduction

Cleaning cloths have been implicated in the spread of bacteria because they provide an ideal environment for bacteria to colonize and grow. When used on a food-contact surface, a cleaning cloth retains food particles that provide nutritional value to bacteria. The bacteria proliferate, resulting in higher concentrations on the cleaning cloth. The cloth then becomes a vehicle of bacterial transfer, either from cloth-to-surface or cloth-to-person, which may contaminate food and result in foodborne illness (Barker & Bloomfield, 2000).

In Australia, an estimated average of 4.2 million cases of foodborne illness occur each year with an estimated cost of over $2.6 billion annually (Food Standards Australia New Zealand, 2002). This situation necessitates the use of preventive sanitizing measures to ensure food hygiene.

The aim of food hygiene is the production and service of safe food. Potential sources of contamination are identified, remedial action can be taken, and the results of the action can be assessed (Angelillo, Viggiani, Greco, & Rito, 2001; McSwane & Linton, 2000). When food has caused food poisoning, it is appropriate to investigate the processing and handling of the product up to the point of consumption. The number of bacteria present and the effectiveness of the sanitizers used should also be assessed. The hygiene status of kitchen surfaces and equipment is an essential element in the production of high-quality, safe foods (Lalla & Dingle, 2004). Hygienic surfaces can be achieved only if the kitchen environment is kept clean and free of bacteria.

It is impossible to eliminate all bacteria from a kitchen environment because of the constant nutritional sources that are present. Bacterial concentrations should be minimized, however, and kept as low as possible so that they can cause neither infection nor harm to the personnel and the public.

A variety of generic sanitizers are used to control bacteriological hazards in the food industry. Quaternary ammonium compounds (QAC) have been predominantly used in the food industry because of their nontainting, noncorrosive, and nontoxic nature with respect to human skin and surfaces (Holah, Taylor, Dawson, & Hall, 2002; Langsrud & Sundheim, 1997). Some species of bacteria, however--namely Staphylococcus species--have shown resistance to QAC sanitizers. Hypochlorites are likely to be the most useful and inexpensive sanitizers for the food industry. They have little taste or smell and have a range of antibacterial activity that is not so limited as that of QAC disinfectants (Hayes, 1992).

The effectiveness of the chemical sanitizers depends on the time of contact, the temperature, and the concentration of the application. Many materials present in food industries have the ability to inactivate chemicals and thus create favorable conditions for bacterial growth. The inactivation of chemicals may occur if organic materials such as food prevent access of the chemical to the bacteria or if the chemical is not used at the recommended concentration. Continuous use of chemicals may lead to bacterial resistance, which allows bacterial growth on both the kitchen surfaces and the equipment (Holah et al., 2002). Therefore, environmentally conscious alternatives for sanitation are thought to reduce the risk of chemical inactivation, bacterial resistance, and chemical exposure to individuals.

The objectives of the study reported here were to monitor the bioburden of Staphylococcus aureus and Escherichia coli on cloths over time and to determine the effectiveness of environmentally conscious alternatives such as use of fiber cloths sanitized with hot water at 75[degrees]C in maintaining concentrations of bacteria on the cloths lower than those found on generic cloths sanitized with hot water at 75[degrees]C or chemical QAC and hypochlorite sanitizers.

Methods

Preparation of Bacterial Suspensions

Gram-positive Staphylococcus aureus ATCC 25923 bacteria were grown in 100 mL of brain heart infusion broth (Amyl Media Pty. Ltd., Dandenong, New South Wales [NSW]) at 37[degrees]C [+ or -] 2[degrees]C for 24 hours. Gram-negative bacteria Escherichia coli UB 1301 bacteria were grown in 100 mL of lauryl tryptose broth (Amyl Media Pty. Ltd., Dandenong, NSW) at 37[degrees]C [+ or -] 2[degrees]C for 18 hours. These culture conditions were found to yield approximately [10.sup.9] CFUs/mL.

Preparation of Neutralizer

The neutralizer was adapted from methods previously described in the Australian Standards (Standards Australia, 1988). Two grams of sodium thiosulphate (Analytical Reagent, Univar, APS Ajax Finechem, Auburn, NSW) and 100 mL of lecithin/Tween 80 emulsion, which contained 2 percent volume per volume (v/v) soybean lecithin (Naytura[TM], Yennora, NSW) in a 3 percent v/v aqueous solution of Tween 80 (Merck Pty. Ltd., Kisyth, Victoria), were added to 1 L of 0.1 percent peptone water. The neutralizer was autoclaved for 20 minutes at 200 kilopascals.

Neutralizer Verification

A suspension of the test organism containing [10.sup.9] CFUs/mL was used to verify the inactivation effect of the neutralizer. A 0.5-mL aliquot of a commercial-grade QAC or hypochlorite sanitizer was added to 9 mL of neutralizer. After 15 seconds, 1 mL of the test organism was added to the solution and shaken on a mechanical shaker for 10 seconds. One-mL and 0.1-mL triplicate samples were plated at 2 minutes, 1 hour, and 6 hours on plate count agar (Oxoid Ltd., Basingstoke, United Kingdom).

[FIGURE 1 OMITTED]

A 1-mL aliquot of the test organism was added to 9.5 mL of neutralizer but no sanitizer. The solution was mixed and triplicate samples were plated as previously described. The plates were incubated at 37[degrees]C [+ or -] 2[degrees]C for 24 hours (Standards Australia, 1992). When the number of colony-forming units per milliliter did not differ between the plates, the neutralizer was considered effective.

Preparation and Inoculation of Surfaces

A food-grade stainless-steel kitchen bench top, Grade 304 (Avesta-Polarit Pty. Ltd., Canning Vale, Western Australia [WA]) was marked with 5 cm X 10 cm squares. The surfaces were sanitized according to a validated cleaning protocol (Moore & Griffith, 2002). Initially the surfaces were disinfected for 5 minutes with a commercial-grade heavy-duty chlorinated sanitizer before being rinsed with boiling water. The surfaces were then thoroughly cleaned with commercial-grade detergent and were rinsed three times with boiling water to remove all detergent residues. All surfaces were left to air-dry for 1 hour at room temperature (Moore & Griffith).

A 0.1-mL aliquot of either S. aureus or E. coli was inoculated and spread evenly onto a 50-[cm.sup.2] surface (Moore & Griffith, 2002). The surfaces were allowed to air-dry for a further hour until no visible liquid remained on the surface.

Microbiological Sampling of Cloths

The fiber cloths that were tested include kitchen fiber cloths and all-purpose fiber cloths (ENJO Pty. Ltd., Willeton, WA). These cloths have longer fibers than do generic cloths and are statically charged to enhance the removal of dirt and dust from surfaces. The generic cloths that were tested included antibacterial cloths and cleaning cloths (Homebrand, Yennora, NSW). All cloths were divided into 310-[cm.sup.2] portions and autoclaved for 20 minutes at 200 kPa.

The cloths were soaked in 75[degrees]C water for 1 minute or a commercial-grade chemical sanitizer such as QAC or hypochlorite for 6 minutes. Sanitizers were applied according to the manufacturers' recommended concentrations. After the 50-[cm.sup.2] surface was wiped four times with a cloth, the cloth was aseptically transferred to 100 mL of 0.1-percent-peptone water and neutralizer. The cloths were stored in sterile, resealable containers at room temperature, and samples were collected from the cloths 2 minutes, 1 hour, and 6 hours after the sanitizing. A 1-mL aliquot was pipetted into 9 mL of 0.1-percent-peptone water, and plates were prepared according to the procedure described above.

[FIGURE 2 OMITTED]

Negative-control assays were performed on surfaces that were not inoculated with bacteria but had been sanitized with a cloth. To conduct positive-control assays, the authors inoculated bacteria onto the surfaces but did not use a cloth to sanitize the surface.

Statistical Analyses

Statistical analyses of the bacterial concentrations were conducted with SPSS 10.0 for Windows (SPSS Inc., Chicago, IL). The concentrations of bacteria were transformed to logarithmic values ([log.sub.10]) to achieve a normal distribution. Mean concentrations were determined, and two-tailed paired t-tests with a confidence interval (CI) of 95 percent were conducted.

Results

Bioburden of Staphylococcus aureus on Cloths

All cloths and sanitizing methods produced a greater-than-5-log reduction in bacterial concentration on the stainless-steel kitchen surfaces 2 minutes after the surfaces were sanitized. The antibacterial cloths sanitized with hot water at 75[degrees]C had the highest concentrations of S. aureus ([log.sub.10] 3.13 CFUs/[m.sup.2]), and the antibacterial cloths sanitized with QAC had the lowest concentrations of S. aureus ([log.sub.10] 0 CFUs/[m.sup.2]) (Figure 1). Concentrations of S. aureus on the kitchen fiber cloths were significantly lower than those on the antibacterial cloths and cleaning cloths sanitized with the different sanitizing methods (p < .050), except for the cleaning cloths sanitized with QAC (p = .172). The all-purpose fiber cloths also had concentrations of S. aureus that were significantly different from concentrations on all the other cloths and sanitizing methods tested in the study (p < .050).

One hour after the stainless-steel kitchen surfaces were sanitized, the highest concentrations of S. aureus were found on the cleaning cloths sanitized with hypochlorite ([log.sub.10] 3.41 CFUs/[m.sup.2]), and the lowest concentrations were found on the antibacterial cloths sanitized with QAC ([log.sub.10] 0.26 CFUs/[m.sup.2]) (Figure 1). The concentrations of S. aureus on the generic cloths sanitized with 75[degrees]C hot water and hypochlorite were significantly higher (p < .050) than the concentrations on the fiber cloths (Figure 1).

Six hours after the stainless-steel kitchen surfaces were sanitized, the highest concentrations of S. aureus were found on the cleaning cloths sanitized with hot water at 75[degrees]C ([log.sub.10] 3.24 CFUs/[m.sup.2]). The lowest concentrations were found on the kitchen fiber cloths sanitized with hot water at 75[degrees]C ([log.sub.10] 0.23 CFUs/[m.sup.2]) (Figure 1). The concentrations of Grampositive bacteria on the kitchen fiber cloths were significantly lower (p = .015) than the concentrations on the all-purpose fiber cloths. The concentrations of S. aureus on the kitchen fiber cloths were not significantly different from the concentrations on the antibacterial cloths sanitized with QAC (p = .084). Furthermore, the concentrations of bacteria on the all-purpose fiber cloths were not significantly different from the concentrations on the antibacterial cloths (p = .739) and cleaning cloths (p = .873) sanitized with QAC (Figure 1). The concentrations of S. aureus on the fiber cloths, however, were significantly lower (p < .050) than those on the generic cloths sanitized with hypochlorite (Figure 1).

Bioburden of Escherichia coli on Cloths

Two minutes after the stainless-steel kitchen surfaces were sanitized, the kitchen fiber cloths ([log.sub.10] 1.32 CFUs/[m.sup.2]) had significantly lower concentrations of Gram-negative E. coli bacteria than did the all-purpose fiber cloths ([log.sub.10] 2.78 CFUs/[m.sup.2]; p = .005). Concentrations of E. coli on the kitchen fiber cloths sanitized with hot water at 75[degrees]C water did not differ significantly from concentrations on the antibacterial cloths (p = .264) and cleaning cloths (p = .193) sanitized with QAC and the antibacterial cloths sanitized with hypochlorite (p = .119). Also, concentrations of E. coli on the all-purpose fiber cloths sanitized with hot water at 75[degrees]C did not differ significantly from concentrations on the antibacterial cloths (p = .148) and cleaning cloths (p = .118) sanitized with hot water at 75[degrees]C or the antibacterial cloths sanitized with hypochlorite (p = .172).

Similarly, 1 hour after the surfaces were sanitized, the concentrations of E. coli on the kitchen fiber cloths sanitized with 75[degrees]C water did not differ significantly from concentrations on the antibacterial cloths (p = .486) and cleaning cloths (p = .195) sanitized with QAC (Figure 2). In addition, the concentrations of E. coli on the all-purpose fiber cloths ([log.sub.10] 2.92 CFUs/[m.sup.2]) did not differ significantly from the concentrations on antibacterial cloths ([log.sub.10] 3.10 CFUs/[m.sup.2]; p = .502) and cleaning cloths ([log.sub.10] 3.07 CFUs/[m.sup.2]; p = .483) sanitized with 75[degrees]C water or from the concentrations on the antibacterial cloths ([log.sub.10] 1.96 CFUs/[m.sup.2]; p = .073) sanitized with hypochlorite (Figure 2).

Six hours after the stainless-steel kitchen surfaces were sanitized, the concentrations of bacteria increased slightly for most of the cloths, except for the all-purpose fiber cloths (Figure 2). The concentrations of E. coli on the kitchen fiber cloths were not significantly different from the concentrations on the antibacterial cloths (p = .900) and cleaning cloths (p = .508) sanitized with QAC. The use of all-purpose fiber cloths did not result in significantly different concentrations of E. coli (p > .050) compared with most of the cloths used with the different sanitizing methods. The cleaning cloths sanitized with hypochlorite ([log.sub.10] 3.38 CFUs/[m.sup.2]) resulted in significantly higher concentrations of E. coli compared with the all-purpose fiber cloths ([log.sub.10] 2.38 CFUs/[m.sup.2]) (Figure 2).

Discussion

Bioburden of Staphylococcus aureus on Cloths

In this study, the sanitizers produced at least a 5-log reduction in bacteria concentrations on the stainless-steel kitchen surfaces. This result indicated that all the sanitizers were effective according to the European standards (Bloomfield, Arthur, Begun, & Patel, 1997). It was also observed that after the stainless-steel kitchen surfaces were sanitized, the concentrations of S. aureus on the kitchen fiber cloths and the generic cloths sanitized with QAC were significantly lower (p > .050) than concentrations on cloths for which different sanitizing methods were used.

In addition, sanitization with hot water at 75[degrees]C or hypochlorite was not as efficient in maintaining lower concentrations of bacteria on the cloths (Figure 1). This result indicates that the sanitizing performance of the fiber cloths was better than that of the generic cloths sanitized with hot water at 75[degrees]C or hypochlorite. The frequent contamination of cloths and the concentrations of bacteria that were present suggest that the cloths are reservoirs and disseminators of bacteria in the kitchen environment (Scott, Bloomfield, & Barlow, 1982). Although most bacteria are unable to survive long periods of time without moisture, they can remain viable for transfer to various surfaces in the kitchen.

In the study reported here, the sanitizing properties of the chemicals are short-lived; after 1 hour, S. aureus concentrations increased on most of the cloths except the fiber cloths (Figure 1). Consequently, an environmentally conscious alternative such as the fiber cloths sanitized with hot water at 75[degrees]C could be used instead of cloths sanitized with chemical sanitizers such as QAC or hypochlorite. The results are consistent with the findings of Rusin, Orosz-Coughlin, and Gerba (1998), who have investigated QAC and hypochlorite sanitizers. The use of QAC resulted in significant reductions in the concentrations of bacteria on the surface and the cloths, while a single application of a hypochlorite sanitizer was short lived.

Bioburden of Escherichia coli on Cloths

After the stainless-steel kitchen surfaces were sanitized, the concentrations of E. coli on the kitchen fiber cloths sanitized with hot water at 75[degrees]C were not significantly higher than concentrations on antibacterial cloths (p = .264) and cleaning cloths (p = .193) sanitized with QAC. By contrast, the concentrations of E. coli found on the generic cloths sanitized with hypochlorite were higher than the concentrations of bacteria found on the kitchen fiber cloths (Figure 2). This result suggests that the sanitizing performance of the kitchen fiber cloths sanitized with hot water at 75[degrees]C was similar to that of the generic cloths sanitized with QAC and hypochlorite. Thus, hot water could be used as an environmentally conscious alternative to the chemical sanitizers.

Hot water is the most reliable, hygienic, and economical method of sanitization (Hobbs & Gilbert, 1981), and it destroys most microorganisms harmful to human health. Cloths used for wet work are heavily contaminated with bacteria unless they are sanitized. Residual bacteria on the cloth may multiply while the cloth is wet, which could entail high concentrations of food-poisoning bacteria on the cloths. The fiber cloths, however, had the ability to maintain lower concentrations of Gram-negative bacteria without the use of harsh and toxic chemicals (Figure 2).

In the study reported here, it was observed that the all-purpose fiber cloths sanitized with hot water at 75[degrees]C had lower concentrations of bacteria than did the generic cloths sanitized with hot water and the cleaning cloths sanitized with hypochlorite (Figure 2). This result suggests that the all-purpose fiber cloths sanitized with hot water at 75[degrees]C provide better sanitizing performance and could be used as an environmentally conscious alternative to generic cloths and chemical hypochlorite when stainless-steel kitchen surfaces are being sanitized in food industry establishments.

When bactericidal agents such as hypochlorites and QAC are being considered, various factors should be taken into account. The concentration of the chemical sanitizers should be controlled, as too much can affect the taste and odor of foods and too little may render the sanitizer ineffective. Chemical sanitizers are destroyed by food debris, and there is no means of determining when the bactericidal action of the sanitizer has been exhausted. Some chemical sanitizers are destructive to silverware, stainless steel, aluminum, and some cloths, and the replacement of kitchen equipment can be costly. Routine use of hot water for sanitizing is more practicable and efficient because kitchen staff may not take the time and trouble to control the strength of the chemicals (Hobbs & Gilbert, 1981; Jay, 1992) and to alternate between QAC and hypochlorite to prevent bacteria from developing resistance to sanitizers (Holah et al., 2002; Langsrud & Sundheim, 1997).

Conclusions

The general standard of hygiene in food industries could be improved through the prevention of cross-contamination by thorough cleaning and sanitizing of surfaces and cloths. Preventing cross-contamination would significantly reduce the concentrations of bacteria in the kitchen environment and ensure the production of safe food. In the study reported here, it was evident that the use of environmentally conscious alternatives such as fiber cloths sanitized with hot water at 75[degrees]C was just as effective as the use of chemical QAC and hypochlorite sanitizers for maintaining low concentrations of both Gram-positive S. aureus and Gram-negative E. coli on the cloths. Therefore, the use of kitchen fiber cloths sanitized with hot water at 75[degrees]C is a reliable, hygienic, and economical method of maintaining kitchen hygiene in food industries.

Corresponding Author: Peter Dingle, Associate Professor and Environmental and Nutritional Toxicologist, School of Environmental Science, Murdoch University, Murdoch, Western Australia, Australia 6150. E-mail: [e-mail].

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Fairuz Lalla

Peter Dingle, Ph.D.

Cedric Cheong